Haemophilia A is a hereditary blood coagulation (clotting) disorder. It is caused by a deficient activity of the plasma protein factor VIII, which affects the clotting property of blood. The standard treatment for Haemophilia A patients is the infusion of factor VIII concentrates to replace the defective clotting factor. However, Haemophilia A patients who develop inhibitors to factor VIII during replacement therapy consequently fail to respond to the above-mentioned therapy and are treated with preparations containing activated coagulation factors (so-called bypassing agents) to achieve haemostasis independently of factor VIII through bypassing mechanisms. Both activated prothrombin complex concentrates (APCC) such as FEIBA (Factor Eight Inhibitor Bypassing Activity—plasma-derived APCC), which triggers the intrinsic or common pathway, and activated factor Vila (rFVIIa) such as NovoSeven (recombinant FVIIa), which is assumed to act via the extrinsic pathway, are favoured options to treat factor VIII inhibitor patients.
No direct monitoring of the drug substance is possible for either treatment regime because the activated components of the bypassing agents interact immediately with proteins of the haemostatic system to induce activation of the clotting cascade. All existing assays measure surrogate markers, which are of limited value for the assessment of the efficacy of the bypassing agent because the specificity and sensitivity depend on the specific assay conditions and because these assays do not give any information on the overall activity status of the haemostatic system.
The ultimate goal of the coagulation cascade is the conversion of prothrombin into thrombin, which then induces clot-formation by activation of fibrinogen. Thus, generation of thrombin is a pivotal function of plasma in haemostasis. Currently there is no routine test that quantitatively assesses the thrombin formation capacity of a plasma sample. Clotting times such as prothrombin time (PTT), activated partial thromboplastin time (aPTT) and thrombin clotting time (TCT) do not reflect the overall thrombin generation because most of the thrombin is formed after the instant of clotting and, therefore, they are insensitive to hypercoagulation and possibly also to hypocoagulation states. The PTT-test measures the clotting time of the extrinsic and common pathway, the aPTT-test measures the clotting time of the intrinsic and common pathway, whereas the TCT-test only measures the clotting time of the common pathway. Hemker et al. (1986. Thromb. Haemost. 56:9-17) were the first to suggest measurement of thrombin generation in patient plasma by assessment of the endogenous thrombin potential. Thrombin generation is a dynamic process. The actual thrombin concentration is dependent on the rate of the activation and inactivation reactions, and thus, reflecting the efficiency of the haemostatic system to control bleeding. Hemker et al. (1995. Thromb. Haemost. 74:134-138) defined the thrombin potential as the overall capacity of plasma to form thrombin after induction of coagulation and proposed the use of this parameter as a sensitive indicator of every form of anticoagulation.
Sultan and Loyer (1993. J. Lab. Clin. Med. 121:444-452) used such a thrombin generation assay for in vitro evaluation of the factor VIII-bypassing activity of activated prothrombin complex concentrates, prothrombin complex concentrates and factor Vila in the plasma of patients with factor VIII inhibitors. There have also been other reports of such assays being used for assessing the efficacy of factor VIII-bypassing agents but, because of their technical complexity, the assays have not disseminated into routine use.
Thrombin generation assays were also described by Turecek at al. (2003. Pathophysiol. Haemost. Thromb. 33:16-22), however, no lyophilized components were used. Moreover, there is the common opinion that lyophilization decreases the activity of the tissue factor.
All the known prior art assays have the disadvantage that components can only be partially dosed before using the assay. Therefore, a lot of preparation steps are necessary to use these assays thereby making the assays uncomfortable and susceptible to mistakes during handling. Moreover, these assays are time-consuming.
It therefore exists a need for a test system which allows the detection and determination of changes, e.g. treatment-dependent changes, in the kinetic of thrombin generation in a sample of a patient's blood or plasma which overcomes the above-mentioned problems.